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应用差速离心和Percoll不连续密度梯度法分离纯化小麦三核期小花线粒体.在裂解液选择、IPG胶条pH值范围、SDS-PAGE胶浓度及蛋白质上样量等方面对线粒体蛋白质双向电泳体系进行探索和优化,确立了一套适用于小麦小花高纯度完整线粒体的分离方法及其蛋白质双向电泳的技术体系.结果表明,采用20%、24%和40%Percoll密度梯度和28%Percoll自形成密度高速离心体系,获得了有活性、高纯度且较完整的线粒体;经TCA-丙酮法提取蛋白,以7 mol/L尿素,2mol/L硫脲,4%CHAPS(W/V),65 mmol/L DTT,0.5%IPG缓冲液(V/V),0.001%溴酚蓝(W/V)裂解液溶解蛋白,采用17 cm,pH 4～7 IPG胶条和11%SDS-PAGE分离胶,上样量为160μg,硝酸银染色法,更适合小麦小花线粒体蛋白质组双向电泳分离.经PDQuest 2DE 8.0.1软件包统计分析,在2-DE图谱上分辨出约150个蛋白点,蛋白点清晰呈圆形,无横条纹干扰,这为利用双向电泳技术在亚细胞水平对线粒体进行蛋白质组学研究与分析奠定了基础,更为进一步分析研究线粒体与雄性不育的关系提供了理论与技术支撑.
The mitochondria were isolated and purified by differential centrifugation and Percoll discontinuous density gradient centrifugation in mitochondrial protein two dimensional gel electrophoresis system in terms of lysate selection, IPG gel pH range, SDS-PAGE gel concentration and protein loading, And established a set of technology system for the separation of high-purity intact mitochondria of wheat flower and its two-dimensional electrophoresis technology.The results showed that using 20%, 24% and 40% Percoll density gradient and 28% Percoll self-formation Density, high-speed centrifugation system to obtain active, high-purity and relatively intact mitochondria. The protein was extracted by TCA-acetone method and was extracted with 7 mol / L urea, 2 mol / L thiourea, 4% CHAPS The lysates were dissolved in 0.5% IPG buffer (V / V) and 0.001% bromophenol blue (W / V) lysate using 17 cm, pH 4-7 IPG strips and 11% SDS- The amount of sample was 160μg, silver nitrate staining was more suitable for wheat mitochondrial two-dimensional electrophoresis separation of proteins by PDQuest 2DE 8.0.1 package statistical analysis, in the 2-DE map resolved about 150 protein spots, protein spots clear Round, no horizontal bar interference, which makes use of two-dimensional electrophoresis in subcellular water Mitochondrial proteomics research and analysis lays the foundation for further analysis of the relationship between a more sterile study of mitochondria and provide theoretical and technical support.